Reversible, long-range radical transfer in E. coli class Ia ribonucleotide reductase

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• Ribonucleotide reductases (RNRs) are crucial enzymes for DNA synthesis, catalyzing nucleotide reduction.
• Class Ia RNRs utilize a stable diferric-tyrosyl radical cofactor for initiating the reaction.
• Radical transfer (RT) over 35 Å via a pathway of redox-active amino acids is proposed in E. coli RNR.

Purpose of the Study:

• To elucidate the mechanistic underpinnings of radical transfer (RT) in class Ia RNRs.
• To investigate the roles of specific tyrosine residues in the RT pathway using unnatural amino acids.
• To characterize the proton-coupled electron transfer (PCET) mechanisms and thermodynamic properties of the RT pathway.

Main Methods:

• Site-selective replacement of pathway tyrosines with unnatural analogues (DOPA, NH2Y, NO2Y, FnYs).
• Application of rapid kinetic techniques, multifrequency EPR, pulsed ENDOR, and 2H-ENDOR spectroscopies.
• Analysis of stable and transient radical intermediates and photoinitiated radical propagation studies.

Main Results:

• Established a specific 35 Å RT pathway involving one stable (Y122•) and three transient (Y356, Y730, Y731) tyrosyl radicals.
• Demonstrated orthogonal PCET in the β2 subunit and co-linear PCET in the α2 subunit.
• Revealed a thermodynamically unfavorable forward RT pathway with decreasing reduction potentials (Y122 < Y356 < Y731 ≈ Y730 ≤ C439).

Conclusions:

• RT is gated by conformational changes occurring at distinct rates (>100 s⁻¹ and ~10 s⁻¹).
• The mechanism involves distinct PCET strategies in different subunits, finely tuned by local environments.
• Understanding RNR radical transfer provides insights into fundamental biological electron transfer processes.